Mammalian cytokine-like polypeptide-10

ABSTRACT

A mammalian cytokine-like polypeptide, called Mammalian Cytokine-like polypeptide-10, (Zcyto 10), polynucleotides encoding the same, antibodies which specifically bind to the polypeptide, and anti-idiotypic antibodies which bind to the antibodies. Zcyto 10 is useful for promoting the healing of wounds and for stimulating the proliferation of platelets.

[0001] The present application is a continuation of co-pending U.S.patent application Ser. No. 10/413,661, filed Apr. 15, 2003,incorporated herein by reference; which is a continuation of U.S. patentapplication Ser. No. 09/199,586, filed Nov. 25, 1998, which claims thebenefit under 35 U.S.C. §119 (e) of U.S. Provisional Application No.60/066,597, filed Nov. 26, 1997.

BACKGROUND OF THE INVENTION

[0002] Proliferation and differentiation of cells of multicellularorganisms are controlled by hormones and polypeptide growth factors.These diffusable molecules allow cells to communicate with each otherand act in concert to form cells and organs, and to repair andregenerate damaged tissue. Examples of hormones and growth factorsinclude the steroid hormones (e.g. estrogen, testosterone), parathyroidhormone, follicle stimulating hormone, the interleukins, plateletderived growth factor (PDGF), epidermal growth factor (EGF),granulocyte-macrophage colony stimulating factor (GM-CSF),erythropoietin (EPO) and calcitonin.

[0003] Hormones and growth factors influence cellular metabolism bybinding to proteins. Proteins may be integral membrane proteins that arelinked to signaling pathways within the cell, such as second messengersystems. Other classes of proteins are soluble molecules.

[0004] Of particular interest are cytokines, molecules that promote theproliferation and/or differentiation of cells. Examples of cytokinesinclude erythropoietin (EPO), which stimulates the development of redblood cells; thrombopoietin (TPO), which stimulates development of cellsof the megakaryocyte lineage; and granulocyte-colony stimulating factor(G-CSF), which stimulates development of neutrophils. These cytokinesare useful in restoring normal blood cell levels in patients sufferingfrom anemia or receiving chemotherapy for cancer. The demonstrated invivo activities of these cytokines illustrates the enormous clinicalpotential of, and need for, other cytokines, cytokine agonists, andcytokine antagonists.

SUMMARY OF THE INVENTION

[0005] The present invention addresses this need by providing a novelpolypeptide and related compositions and methods. Within one aspect, thepresent invention provides an isolated polynucleotide encoding amammalian four alpha helix cytokine termed Zcyto 10. The human Zcyto 10polypeptide is comprised of a sequence of 176 amino acids with theinitial Met as shown in SEQ ID NO:1 and SEQ ID NO:2. It is believed thatamino residues 1-24 are signal sequence, and the mature Zcyto10polypeptide is represented by the amino acid sequence comprised ofresidues 25, a leucine, through amino acid residue 176, a glutamic acidresidue, also defined by SEQ ID NO:12. Another embodiment of the presentinvention is defined by the sequences of SEQ ID NO: 3 and SEQ ID NO: 4.The polypeptide of SEQ ID NO: 4 is comprised of 151 amino acid residueswherein amino acids 1-24 comprise a signal sequence and the maturesequence is comprised of amino acid residues 25, a leucine, throughamino acid 151 a glutamic acid, also defined by SEQ ID NO:13. Anotheractive variant is comprised of amino acid residues 33, a cysteine,through amino acid residue 176 of SEQ ID NO:2. This variant is alsodefined by SEQ ID NO:26.

[0006] Mouse Zcyto 10 is also a polypeptide comprised of 176 amino acidresidues as defined by SEQ ID NOs: 18 and 19. Mouse Zcyto10 has a signalsequence extending from amino acid residue 1, a methionine, extending toand including amino acid residue 24, a glycine of SEQ ID NO:19. Thus,the mature mouse Zcyto10 extends from amino acid residue 25, a leucine,to and including amino acid residue 176 a leucine of SEQ ID NO:19, alsodefined by SEQ ID NO:20. Another active variant is believed to extendfrom amino acid 33, a cysteine, through amino acid 176, of SEQ ID NO:19.This variant is also defined by SEQ ID NO:25. Within an additionalembodiment, the polypeptide further comprises an affinity tag.

[0007] A variant of mouse Zcyto10 is defined by SEQ ID NOs: 33 and 34.This variant is 154 amino acid residues in length and has a signalsequence extending from amino acid residue 1, a methionine, to andincluding amino acid residue 24, a glycine, of SEQ ID NO:34. Thus, themature sequence extends from amino acid residue 25, a leucine, to andincluding amino acid residue 154, a leucine, of SEQ ID NO:34. The maturesequence is also defined by SEQ ID NO:35.

[0008] Within a second aspect of the invention there is provided anexpression vector comprising (a) a transcription promoter; (b) a DNAsegment encoding Zcyto10 polypeptide, and (c) a transcriptionterminator, wherein the promoter, DNA segment, and terminator areoperably linked.

[0009] Within a third aspect of the invention there is provided acultured eukaryotic or prokaryotic cell into which has been introducedan expression vector as disclosed above, wherein said cell expresses apolypeptide encoded by the DNA segment.

[0010] Within a further aspect of the invention there is provided achimeric polypeptide consisting essentially of a first portion and asecond portion joined by a peptide bond. The first portion of thechimeric polypeptide consists essentially of (a) a Zcyto10 polypeptideas shown in SEQ ID NO: 2 (b) allelic variants of SEQ ID NO:2, SEQ IDNO:4, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:19, SEQ ID NO:20, SEQ. IDNO:25, SEQ ID NO:26 SEQ ID NO:34 or SEQ ID NO:35; and (c) proteinpolypeptides that are at least 90% identical to (a) or (b). The secondportion of the chimeric polypeptide consists essentially of anotherpolypeptide such as an affinity tag. Within one embodiment the affinitytag is an immunoglobulin Fc polypeptide. The invention also providesexpression vectors encoding the chimeric polypeptides and host cellstransfected to produce the chimeric polypeptides.

[0011] Within an additional aspect of the invention there is provided anantibody that specifically binds to a Zcyto10 polypeptide as disclosedabove, and also an anti-idiotypic antibody which neutralizes theantibody to a Zcyto10 polypeptide. Within another aspect of the presentinvention there is provided a pharmaceutical composition comprisingpurified Zcyto10 polypeptide in combination with a pharmaceuticallyacceptable vehicle. Such compositions may be useful for modulating ofcell proliferation, cell differentiation or cytokine production in theprevention or treatment of conditions characterized by improper cellproliferation, cell differentiation or cytokine production, as arefurther discussed herein. More specifically, Zcyto10 polypeptide may beuseful in the prevention or treatment of autoimmune diseases byinhibiting a cellular immune response. Autoimmune diseases which may beamenable to Zcyto10 treatment include IDDM, multiple sclerosis,rheumatoid arthritis and the like. Also, Zcyto10 polypeptides of thepresent invention may be useful in inhibiting cancer cell growth orproliferation.

[0012] Zcyto10 polypeptides of the present invention may also stimulatethe immune system to better combat microbial or viral infections. Inparticular, Zcyto10 can be administered systemically to increaseplatelet production by an individual. Moreover, Zcyto10 polypeptides ofthe present invention may be used in trachea- specific ortracheobronchial-specific applications, such as in the maintenance orwound repair of the tracheobronchial epithelium or cells underlying thesame, in regulating mucous production or mucocilary clearance of debrisor in treatment of asthma, bronchitis or other diseases of thetracheobronchial tract. It may also enhance wound healing and promoteregeneration of affected tissues which may be especially useful in thetreatment of periodontal disease. Furthermore, Zcyto10 polypeptides canbe used to treat skin conditions such as psoriasis, eczema and dry skinin general.

[0013] An additional embodiment of the present invention relates to apeptide or polypeptide which has the amino acid sequence of anepitope-bearing portion of a Zcyto10 polypeptide having an amino acidsequence described above. Peptides or polypeptides having the amino acidsequence of an epitope-bearing portion of a Zcyto10 polypeptide of thepresent invention include portions of such polypeptides with at leastnine, preferably at least 15 and more preferably at least 30 to 50 aminoacids, although epitope-bearing polypeptides of any length up to andincluding the entire amino acid sequence of a polypeptide of the presentinvention described above are also included in the present invention.Also claimed are any of these polypeptides that are fused to anotherpolypeptide or carrier molecule. Such epitope variants include but arenot limited to SEQ ID NOs: 25-32. Antibodies produced from theseepitope-bearing portions of Zcyto10 can be used in purifying Zcyto10from cell culture medium.

[0014] These and other aspects of the invention will become evident uponreference to the following detailed description and the attacheddrawing.

DETAILED DESCRIPTION OF THE INVENTION

[0015] The teachings of all the references cited herein are incorporatedin their entirety by reference.

[0016] Prior to setting forth the invention in detail, it may be helpfulto the understanding thereof to define the following terms:

[0017] The term “affinity tag” is used herein to denote a polypeptidesegment that can be attached to a second polypeptide to provide forpurification or detection of the second polypeptide or provide sites forattachment of the second polypeptide to a substrate. In principal, anypeptide or protein for which an antibody or other specific binding agentis available can be used as an affinity tag. Affinity tags include apoly-histidine tract, protein A, Nilsson et al., EMBO J. 4:1075 (1985);Nilsson et al., Methods Enzymol. 198:3 (1991), glutathione Stransferase, Smith and Johnson, Gene 67:31 (1988), Glu-Glu affinity tag,Grussenmeyer et al., Proc. Natl. Acad. Sci. USA 82:7952-4 (1985),substance P, Flag™ peptide, Hopp et al., Biotechnology 6:1204-1210(1988), streptavidin binding peptide, or other antigenic epitope orbinding domain. See, in general, Ford et al., Protein Expression andPurification 2: 95-107 (1991). DNAs encoding affinity tags are availablefrom commercial suppliers (e.g., Pharmacia Biotech, Piscataway, N.J.).

[0018] The term “allelic variant” is used herein to denote any of two ormore alternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inphenotypic polymorphism within populations. Gene mutations can be silent(no change in the encoded polypeptide) or may encode polypeptides havingaltered amino acid sequence. The term allelic variant is also usedherein to denote a protein encoded by an allelic variant of a gene.

[0019] The terms “amino-terminal” and “carboxyl-terminal” are usedherein to denote positions within polypeptides. Where the contextallows, these terms are used with reference to a particular sequence orportion of a polypeptide to denote proximity or relative position. Forexample, a certain sequence positioned carboxyl-terminal to a referencesequence within a polypeptide is located proximal to the carboxylterminus of the reference sequence, but is not necessarily at thecarboxyl terminus of the complete polypeptide.

[0020] The term “complement/anti-complement pair” denotes non-identicalmoieties that form a non-covalently associated, stable pair underappropriate conditions. For instance, biotin and avidin (orstreptavidin) are prototypical members of a complement/anti-complementpair. Other exemplary complement/anti-complement pairs includereceptor/ligand pairs, antibody/antigen (or hapten or epitope) pairs,sense/antisense polynucleotide pairs, and the like. Where subsequentdissociation of the complement/anti-complement pair is desirable, thecomplement/anti-complement pair preferably has a binding affinity of<10⁹ M⁻¹.

[0021] The term “complements of a polynucleotide molecule” is apolynucleotide molecule having a complementary base sequence and reverseorientation as compared to a reference sequence. For example, thesequence 5′ ATGCACGGG 3′ is complementary to 5′ CCCGTGCAT 3′.

[0022] The term “contig” denotes a polynucleotide that has a contiguousstretch of identical or complementary sequence to anotherpolynucleotide. Contiguous sequences are said to “overlap” a givenstretch of polynucleotide sequence either in their entirety or along apartial stretch of the polynucleotide. For example, representativecontigs to the polynucleotide sequence 5′-ATGGCTTAGCTT-3′ are5′-TAGCTTgagtct-3′ and 3′-gtcgacTACCGA-5′.

[0023] The term “degenerate nucleotide sequence” denotes a sequence ofnucleotides that includes one or more degenerate codons (as compared toa reference polynucleotide molecule that encodes a polypeptide).Degenerate codons contain different triplets of nucleotides, but encodethe same amino acid residue (i.e., GAU and GAC triplets each encodeAsp).

[0024] The term “expression vector” is used to denote a DNA molecule,linear or circular, that comprises a segment encoding a polypeptide ofinterest operably linked to additional segments that provide for itstranscription. Such additional segments include promoter and terminatorsequences, and may also include one or more origins of replication, oneor more selectable markers, an enhancer, a polyadenylation signal, etc.Expression vectors are generally derived from plasmid or viral DNA, ormay contain elements of both.

[0025] The term “isolated”, when applied to a polynucleotide, denotesthat the polynucleotide has been removed from its natural genetic milieuand is thus free of other extraneous or unwanted coding sequences, andis in a form suitable for use within genetically engineered proteinproduction systems. Such isolated molecules are those that are separatedfrom their natural environment and include cDNA and genomic clones.Isolated DNA molecules of the present invention are free of other geneswith which they are ordinarily associated, but may include naturallyoccurring 5′ and 3′ untranslated regions such as promoters andterminators. The identification of associated regions will be evident toone of ordinary skill in the art (see for example, Dynan and Tijan,Nature 316:774-78 (1985).

[0026] An “isolated” polypeptide or protein is a polypeptide or proteinthat is found in a condition other than its native environment, such asapart from blood and animal tissue. In a preferred form, the isolatedpolypeptide is substantially free of other polypeptides, particularlyother polypeptides of animal origin. It is preferred to provide thepolypeptides in a highly purified form, i.e. greater than 95% pure, morepreferably greater than 99% pure. When used in this context, the term“isolated” does not exclude the presence of the same polypeptide inalternative physical forms, such as dimers or alternatively glycosylatedor derivatized forms.

[0027] The term “operably linked”, when referring to DNA segments,indicates that the segments are arranged so that they function inconcert for their intended purposes, e.g., transcription initiates inthe promoter and proceeds through the coding segment to the terminator.

[0028] The term “ortholog” denotes a polypeptide or protein obtainedfrom one species that is the functional counterpart of a polypeptide orprotein from a different species. Sequence differences among orthologsare the result of speciation.

[0029] “Paralogs” are distinct but structurally related proteins made byan organism. Paralogs are believed to arise through gene duplication.For example, a-globin, b-globin, and myoglobin are paralogs of eachother.

[0030] A “polynucleotide” is a single- or double-stranded polymer ofdeoxyribonucleotide or ribonucleotide bases read from the 5′ to the 3′end. Polynucleotides include RNA and DNA, and may be isolated fromnatural sources, synthesized in vitro, or prepared from a combination ofnatural and synthetic molecules. Sizes of polynucleotides are expressedas base pairs (abbreviated “bp”), nucleotides (“nt”), or kilobases(“kb”). Where the context allows, the latter two terms may describepolynucleotides that are single-stranded or double-stranded. When theterm is applied to double-stranded molecules it is used to denoteoverall length and will be understood to be equivalent to the term “basepairs”. It will be recognized by those skilled in the art that the twostrands of a double-stranded polynucleotide may differ slightly inlength and that the ends thereof may be staggered as a result ofenzymatic cleavage; thus all nucleotides within a double-strandedpolynucleotide molecule may not be paired. Such unpaired ends will ingeneral not exceed 20 nt in length.

[0031] A “polypeptide” is a polymer of amino acid residues joined bypeptide bonds, whether produced naturally or synthetically. Polypeptidesof less than about 10 amino acid residues are commonly referred to as“peptides”.

[0032] The term “promoter” is used herein for its art-recognized meaningto denote a portion of a gene containing DNA sequences that provide forthe binding of 15 RNA polymerase and initiation of transcription.Promoter sequences are commonly, but not always, found in the 5′non-coding regions of genes.

[0033] A “protein” is a macromolecule comprising one or more polypeptidechains. A protein may also comprise non-peptidic components, such ascarbohydrate groups. Carbohydrates and other non-peptidic substituentsmay be added to a protein by the cell in which the protein is produced,and will vary with the type of cell. Proteins are defined herein interms of their amino acid backbone structures; substituents such ascarbohydrate groups are generally not specified, but may be presentnonetheless.

[0034] The term “receptor” denotes a cell-associated protein that bindsto a bioactive molecule (i.e., a ligand) and mediates the effect of theligand on the cell. Membrane-bound receptors are characterized by amulti-domain structure comprising an extracellular ligand-binding domainand an intracellular effector domain that is typically involved insignal transduction. Binding of ligand to receptor results in aconformational change in the receptor that causes an interaction betweenthe effector domain and other molecule(s) in the cell. This interactionin turn leads to an alteration in the metabolism of the cell. Metabolicevents that are linked to receptor-ligand interactions include genetranscription, phosphorylation, dephosphorylation, increases in cyclicAMP production, mobilization of cellular calcium, mobilization ofmembrane lipids, cell adhesion, hydrolysis of inositol lipids andhydrolysis of phospholipids. In general, receptors can be membranebound, cytosolic or nuclear; monomeric (e.g., thyroid stimulatinghormone receptor, beta-adrenergic receptor) or multimeric (e.g., PDGFreceptor, growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSFreceptor, erythropoietin receptor and IL-6 receptor).

[0035] The term “secretory signal sequence” denotes a DNA sequence thatencodes a polypeptide (a “secretory peptide”) that, as a component of alarger polypeptide, directs the larger polypeptide through a secretorypathway of a cell in which it is synthesized. The larger polypeptide iscommonly cleaved to remove the secretory peptide during transit throughthe secretory pathway.

[0036] The term “splice variant” is used herein to denote alternativeforms of RNA transcribed from a gene. Splice variation arises naturallythrough use of alternative splicing sites within a transcribed RNAmolecule, or less commonly between separately transcribed RNA molecules,and may result in several mRNAs transcribed from the same gene. Splicevariants may encode polypeptides having altered amino acid sequence. Theterm splice variant is also used herein to denote a protein encoded by asplice variant of an mRNA transcribed from a gene.

[0037] Molecular weights and lengths of polymers determined by impreciseanalytical methods (e.g., gel electrophoresis) will be understood to beapproximate values. When such a value is expressed as “about” X or“approximately” X, the stated value of X will be understood to beaccurate to ±10%.

[0038] It is believed that Zcyto10 is of a member of the IL-10 subfamilyof cytokines. Other members of this group include MDA-7, IL-19, and KFF.Conserved amino acids in the helix D of Zcyto10 can be used as a tool toidentify new family members. Helix D is the most highly conserved havingabout 32% identity with the helix D of IL-10. For instance, reversetranscription-polymerase chain reaction (RT-PCR) can be used to amplifysequences encoding the conserved [the domain, region or motif fromabove] from RNA obtained from a variety of tissue sources or cell lines.In particular, highly degenerate primers designed from the Zcyto10sequences are useful for this purpose.

[0039] Within preferred embodiments of the invention the isolatedpolynucleotides will hybridize to similar sized regions of SEQ ID NO: 1,SEQ ID NO:3, SEQ ID NO: 18, SEQ ID NO:33 or a sequence complementarythereto, under stringent conditions. In general, stringent conditionsare selected to be about 5° C. lower than the thermal melting point(T_(m)) for the specific sequence at a defined ionic strength and pH.The T_(m) is the temperature (under defined ionic strength and pH) atwhich 50% of the target sequence hybridizes to a perfectly matchedprobe. Typical stringent conditions are those in which the saltconcentration is about 0.02 M or less at pH 7 and the temperature is atleast about 60° C. As previously noted, the isolated polynucleotides ofthe present invention include DNA and RNA. Methods for isolating DNA andRNA are well known in the art. Total RNA can be prepared using guanidineHCl extraction followed by isolation by centrifugation in a CsClgradient [Chirgwin et al., Biochemistry 18:52-94, (1979)]. Poly (A)⁺ RNAis prepared from total RNA using the method of Aviv and Leder, Proc.Natl. Acad. Sci. USA 69:1408-1412 (1972). Complementary DNA (cDNA) isprepared from poly(A)⁺ RNA using known methods. Polynucleotides encodingZcyto10 polypeptides are then identified and isolated by, for example,hybridization or PCR.

[0040] Additionally, the polynucleotides of the present invention can besynthesized using a DNA synthesizer. Currently the method of choice isthe phosphoramidite method. If chemically synthesized double strandedDNA is required for an application such as the synthesis of a gene or agene fragment, then each complementary strand is made separately. Theproduction of short genes (60 to 80 bp) is technically straightforwardand can be accomplished by synthesizing the complementary strands andthen annealing them. For the production of longer genes (>300 bp),however, special strategies must be invoked, because the couplingefficiency of each cycle during chemical DNA synthesis is seldom 100%.To overcome this problem, synthetic genes (double-stranded) areassembled in modular form from single-stranded fragments that are from20 to 100 nucleotides in length. See Glick, Bernard R. and Jack J.Pasternak, Molecular Biotechnology, Principles & Applications ofRecombinant DNA, (ASM Press, Washington, D.C. 1994), Itakura, K. et al.Synthesis and use of synthetic oligonucleotides. Annu. Rev. Biochem. 53:323-356 (1984), and Climie, S. et al. Chemical synthesis of thethymidylate synthase gene. Proc. Natl. Acad. Sci. USA 87 :633-637(1990).

[0041] Those skilled in the art will recognize that the sequencesdisclosed in SEQ ID NOs:1, 2, 3 and 4 represent a two alleles of thehuman, and SEQ ID NOs:18, 19, 33 and 34 represent two alleles of themouse. Additional allelic variants of these sequences can be cloned byprobing cDNA or genomic libraries from different individuals accordingto standard procedures. Allelic variants of this sequence can be clonedby probing cDNA or genomic libraries from different individualsaccording to standard procedures. Allelic variants of the DNA sequenceshown in SEQ ID NO:1, including those containing silent mutations andthose in which mutations result in amino acid sequence changes, arewithin the scope of the present invention, as are proteins which areallelic variants of SEQ ID NO:2. cDNAs generated from alternativelyspliced mRNAs, which retain the properties of the Zcyto10 polypeptideare included within the scope of the present invention, as arepolypeptides encoded by such cDNAs and mRNAs. Allelic variants andsplice variants of these sequences can be cloned by probing cDNA orgenomic libraries from different individuals or tissues according tostandard procedures known in the art.

[0042] The present invention further provides counterpart proteins andpolynucleotides from other species (“species orthologs”). Of particularinterest are Zcyto10 polypeptides from other mammalian species,including murine, porcine, ovine, bovine, canine, feline, equine, andother primates. Species orthologs of the human Zcyto10 protein can becloned using information and compositions provided by the presentinvention in combination with conventional cloning techniques. Forexample, a cDNA can be cloned using mRNA obtained from a tissue or celltype that expresses the protein. Suitable sources of mRNA can beidentified by probing Northern blots with probes designed from thesequences disclosed herein. A library is then prepared from mRNA of apositive tissue or cell line. A protein-encoding cDNA can then beisolated by a variety of methods, such as by probing with a complete orpartial human or mouse cDNA or with one or more sets of degenerateprobes based on the disclosed sequences. A cDNA can also be cloned usingthe polymerase chain reaction, or PCR (Mullis et al. U.S. Pat. No.4,683,202), using primers designed from the sequences disclosed herein.Within an additional method, the cDNA library can be used to transformor transfect host cells, and expression of the cDNA of interest can bedetected with an antibody to the protein. Similar techniques can also beapplied to the isolation of genomic clones. As used and claimed, thelanguage “an isolated polynucleotide which encodes a polypeptide, saidpolynucleotide being defined by SEQ ID NOs: 2, 4 12, 13, 19, 20, 25, 26,34 and 35” includes all allelic variants and species orthologs of thesepolypeptides.

[0043] The present invention also provides isolated protein polypeptidesthat are substantially identical to the protein polypeptides of SEQ IDNO: 2 and its species orthologs. By “isolated” is meant a protein orpolypeptide that is found in a condition other than its nativeenvironment, such as apart from blood and animal tissue. In a preferredform, the isolated polypeptide is substantially free of otherpolypeptides, particularly other polypeptides of animal origin. It ispreferred to provide the polypeptides in a highly purified form, i.e.greater than 95% pure, more preferably greater than 99% pure. The term“substantially identical” is used herein to denote polypeptides having50%, preferably 60%, more preferably at least 80%, sequence identity tothe sequence shown in SEQ ID NOs: 2, 4 12, 13, 19, 20, 25, 26, 34 and35, or their species orthologs. Such polypeptides will more preferablybe at least 90% identical, and most preferably 95% or more identical toSEQ ID NO:2, or its species orthologs. Percent sequence identity isdetermined by conventional methods. See, for example, Altschul et. al.,Bull. Math. Bio. 48: 603-616 (1986) and Henikoff and Henikoff, Proc.Natl. Acad. Sci. USA 89:10915-10919 (1992). Briefly, two amino acidsequences are aligned to optimize the alignment scores using a gapopening penalty of 10, a gap extension penalty of 1, and the “blossom62” scoring matrix of Henikoff and Henikoff (ibid.) as shown in Table 1(amino acids are indicated by the standard one-letter codes). Thepercent identity is then calculated as:$\frac{{Total}\quad {number}\quad {of}\quad {identical}\quad {matches}}{\begin{matrix}\lbrack {{length}\quad {of}\quad {the}\quad {longer}\quad {sequence}\quad {plus}\quad {the}\quad {number}\quad {of}}  \\{{{gaps}\quad {introduced}\quad {into}\quad {the}\quad {longer}\quad {sequence}\quad {in}\quad {order}}\quad} \\ {{to}\quad {align}\quad {the}\quad {two}\quad {sequences}} \rbrack\end{matrix}} \times 100$

TABLE 1 A R N D C Q E G H I L K M F P S T W Y V A 4 R −1 5 N −2 0 6 D −2−2 1 6 C 0 −3 −3 −3 9 Q −1 1 0 0 −3 5 E −1 0 0 2 −4 2 5 G 0 −2 0 −1 −3−2 −2 6 H −2 0 1 −1 −3 0 0 −2 8 I −1 −3 −3 −3 −1 −3 −3 −4 −3 4 L −1 −2−3 −4 −1 −2 −3 −4 −3 2 4 K −1 2 0 −1 −3 1 1 −2 −1 −3 −2 5 M −1 −1 −2 −3−1 0 −2 −3 −2 1 2 −1 5 F −2 −3 −3 −3 −2 −3 −3 −3 −1 0 0 −3 0 6 P −1 −2−2 −1 −3 −1 −1 −2 −2 −3 −3 −1 −2 −4 7 S 1 −1 1 0 −1 0 0 0 −1 −2 −2 0 −1−2 −1 4 T 0 −1 0 −1 −1 −1 −1 −2 −2 −1 −1 −1 −1 −2 −1 1 5 W −3 −3 −4 −4−2 −2 −3 −2 −2 −3 −2 −3 −1 1 −4 −3 −2 11 Y −2 −2 −2 −3 −2 −1 −2 −3 2 −1−1 −2 −1 3 −3 −2 −2 2 7 V 0 −3 −3 −3 −1 −2 −2 −3 −3 3 1 −2 1 −1 −2 −2 0−3 −1 4

[0044] Sequence identity of polynucleotide molecules is determined bysimilar methods using a ratio as disclosed above.

[0045] Variant Zcyto10 polypeptides or substantially identical proteinsand polypeptides are characterized as having one or more amino acidsubstitutions, deletions or additions. These changes are preferably of aminor nature, that is conservative amino acid substitutions (see Table2) and other substitutions that do not significantly affect the foldingor activity of the protein or polypeptide; small deletions, typically ofone to about 30 amino acids; and small amino- or carboxyl-terminalextensions, such as an amino-terminal methionine residue, a small linkerpeptide of up to about 20-25 residues, or a small extension thatfacilitates purification (an affinity tag), such as a poly-histidinetract, protein A, Nilsson et al., EMBO J. 4:1075 (1985); Nilsson et al.,Methods Enzymol. 198:3 (1991), glutathione S transferase, Smith andJohnson, Gene 67:31 (1988), or other antigenic epitope or bindingdomain. See, in general Ford et al., Protein Expression and Purification2: 95-107 (1991). DNAs encoding affinity tags are available fromcommercial suppliers (e.g., Pharmacia Biotech, Piscataway, N.J.). TABLE2 Conservative amino acid substitutions Basic: arginine lysine histidineAcidic: glutamic acid aspartic acid Polar: glutamine asparagineHydrophobic: leucine isoleucine valine Aromatic: phenylalaninetryptophan tyrosine Small: glycine alanine serine threonine methionine

[0046] The present invention further provides a variety of otherpolypeptide fusions [and related multimeric proteins comprising one ormore polypeptide fusions]. For example, a Zcyto10 polypeptide can beprepared as a fusion to a dimerizing protein as disclosed in U.S. Pat.Nos. 5,155,027 and 5,567,584. Preferred dimerizing proteins in thisregard include immunoglobulin constant region domains.Immunoglobulin-Zcyto10 polypeptide fusions can be expressed ingenetically engineered cells [to produce a variety of multimeric Zcyto10analogs]. Auxiliary domains can be fused to Zcyto10 polypeptides totarget them to specific cells, tissues, or macromolecules (e.g.,collagen). For example, a Zcyto10 polypeptide or protein could betargeted to a predetermined cell type by fusing a polypeptide to aligand that specifically binds to a receptor on the surface of thetarget cell. In this way, polypeptides and proteins can be targeted fortherapeutic or diagnostic purposes. A Zcyto10 polypeptide can be fusedto two or more moieties, such as an affinity tag for purification and atargeting domain. Polypeptide fusions can also comprise one or morecleavage sites, particularly between domains. See, Tuan et al.,Connective Tissue Research 34:1-9 (1996).

[0047] The proteins of the present invention can also comprisenon-naturally occurring amino acid residues. Non-naturally occurringamino acids include, without limitation, trans-3-methylproline,2,4-methanoproline, cis-4-hydroxyproline, trans-4-hydroxyproline,N-methylglycine, allo-threonine, methylthreonine, hydroxyethylcysteine,hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid,thiazolidine carboxylic acid, dehydroproline, 3- and 4-methylproline,3,3-dimethylproline, tert-leucine, norvaline, 2-azaphenylalanine,3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine.Several methods are known in the art for incorporating non-naturallyoccurring amino acid residues into proteins. For example, an in vitrosystem can be employed wherein nonsense mutations are suppressed usingchemically aminoacylated suppressor tRNAs. Methods for synthesizingamino acids and aminoacylating tRNA are known in the art. Essentialamino acids in the polypeptides of the present invention can beidentified according to procedures known in the art, such assite-directed mutagenesis or alanine-scanning mutagenesis [Cunninghamand Wells, Science 244: 1081-1085 (1989)]; Bass et al., Proc. Natl.Acad. Sci. USA 88:4498-4502 (1991). In the latter technique, singlealanine mutations are introduced at every residue in the molecule, andthe resultant mutant molecules are tested for biological activity (e.g.,ligand binding and signal transduction) to identify amino acid residuesthat are critical to the activity of the molecule. Sites of ligand-protein interaction can also be determined by analysis of crystalstructure as determined by such techniques as nuclear magneticresonance, crystallography or photoaffinity labeling. See, for example,de Vos et al., Science 255:306-312 (1992); Smith et al., J. Mol. Biol.224:899-904 (1992); Wlodaver et al., FEBS Lett. 309:59-64 (1992). Theidentities of essential amino acids can also be inferred from analysisof homologies with related proteins.

[0048] Multiple amino acid substitutions can be made and tested usingknown methods of mutagenesis and screening, such as those disclosed byReidhaar-Olson and Sauer, Science 241:53-57 (1988) or Bowie and SauerProc. Natl. Acad. Sci. USA 86:2152-2156 (1989). Briefly, these authorsdisclose methods for simultaneously randomizing two or more positions ina polypeptide, selecting for functional polypeptide, and then sequencingthe mutagenized polypeptides to determine the spectrum of allowablesubstitutions at each position. Other methods that can be used includephage display (e.g., Lowman et al., Biochem. 30:10832-10837 (1991);Ladner et al., U.S. Pat. No. 5,223,409; Huse, WIPO Publication WO92/06204) and region-directed mutagenesis, Derbyshire et al., Gene46:145 (1986); Ner et al., DNA 7:127 (1988).

[0049] Mutagenesis methods as disclosed above can be combined withhigh-throughput screening methods to detect activity of cloned,mutagenized proteins in host cells. Preferred assays in this regardinclude cell proliferation assays and biosensor-based ligand-bindingassays, which are described below. Mutagenized DNA molecules that encodeactive proteins or portions thereof (e.g., ligand-binding fragments) canbe recovered from the host cells and rapidly sequenced using modemequipment. These methods allow the rapid determination of the importanceof individual amino acid residues in a polypeptide of interest, and canbe applied to polypeptides of unknown structure.

[0050] Using the methods discussed above, one of ordinary skill in theart can prepare a variety of polypeptides that are substantiallyidentical to SEQ ID NOs: 2, 4 12, 13, 19, 20, 25, 26, 34 and 35 orallelic variants thereof and retain the properties of the wild-typeprotein. As expressed and claimed herein the language, “a polypeptide asdefined by SEQ ID NO: 2” includes all allelic variants and speciesorthologs of the polypeptide.

[0051] The protein polypeptides of the present invention, includingfull-length proteins, protein fragments (e.g. ligand-binding fragments),and fusion polypeptides can be produced in genetically engineered hostcells according to conventional techniques. Suitable host cells arethose cell types that can be transformed or transfected with exogenousDNA and grown in culture, and include bacteria, fungal cells, andcultured higher eukaryotic cells. Eukaryotic cells, particularlycultured cells of multicellular organisms, are preferred. Techniques formanipulating cloned DNA molecules and introducing exogenous DNA into avariety of host cells are disclosed by Sambrook et al., MolecularCloning: A Laboratory Manual, 2nd ed. (Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989), and Ausubel et al., ibid.

[0052] Polynucleotides, generally a cDNA sequence, of the presentinvention encode the above-described polypeptides. A DNA sequence whichencodes a polypeptide of the present invention is comprised of a seriesof codons, each amino acid residue of the polypeptide being encoded by acodon and each codon being comprised of three nucleotides. The aminoacid residues are encoded by their respective codons as follows. Alanine(Ala) is encoded by GCA, GCC, GCG or GCT; Cysteine (Cys) is encoded byTGC or TGT; Aspartic acid (Asp) is encoded by GAC or GAT; Glutamic acid(Glu) is encoded by GAA or GAG; Phenylalanine (Phe) is encoded by TTC orTTT; Glycine (Gly) is encoded by GGA, GGC, GGG or GGT; Histidine (His)is encoded by CAC or CAT; Isoleucine (Ile) is encoded by ATA, ATC orATT; Lysine (Lys) is encoded by AAA, or AAG; Leucine (Leu) is encoded byTTA, TTG, CTA, CTC, CTG or CTT; Methionine (Met) is encoded by ATG;Asparagine (Asn) is encoded by AAC or AAT; Proline (Pro) is encoded byCCA, CCC, CCG or CCT; Glutamine (Gln) is encoded by CAA or CAG; Arginine(Arg) is encoded by AGA, AGG, CGA, CGC, CGG or CGT; Serine (Ser) isencoded by AGC, AGT, TCA, TCC, TCG or TCT; Threonine (Thr) is encoded byACA, ACC, ACG or ACT; Valine (Val) is encoded by GTA, GTC, GTG or GTT;Tryptophan (Trp) is encoded by TGG; and Tyrosine (Tyr) is encoded by TACor TAT.

[0053] It is to be recognized that according to the present invention,when a cDNA is claimed as described above, it is understood that what isclaimed are both the sense strand, the anti-sense strand, and the DNA asdouble-stranded having both the sense and anti-sense strand annealedtogether by their respective hydrogen bonds. Also claimed is themessenger RNA (mRNA) which encodes the polypeptides of the presentinvention, and which mRNA is encoded by the above-described cDNA. Amessenger RNA (mRNA) will encode a polypeptide using the same codons asthose defined above, with the exception that each thymine nucleotide (T)is replaced by a uracil nucleotide (U).

[0054] In general, a DNA sequence encoding a Zcyto10 polypeptide isoperably linked to other genetic elements required for its expression,generally including a transcription promoter and terminator, within anexpression vector. The vector will also commonly contain one ormore-selectable markers and one or more origins of replication, althoughthose skilled in the art will recognize that within certain systemsselectable markers may be provided on separate vectors, and replicationof the exogenous DNA may be provided by integration into the host cellgenome. Selection of promoters, terminators, selectable markers, vectorsand other elements is a matter of routine design within the level ofordinary skill in the art. Many such elements are described in theliterature and are available through commercial suppliers.

[0055] To direct a Zcyto10 polypeptide into the secretory pathway of ahost cell, a secretory signal sequence (also known as a leader sequence,prepro sequence or pre sequence) is provided in the expression vector.The secretory signal sequence may be that of the protein, or may bederived from another secreted protein (e.g., t-PA) or synthesized denovo. The secretory signal sequence is joined to the Zcyto10 DNAsequence in the correct reading frame. Secretory signal sequences arecommonly positioned 5′ to the DNA sequence encoding the polypeptide ofinterest, although certain signal sequences may be positioned elsewherein the DNA sequence of interest (see, e.g., Welch et al., U.S. Pat. No.5,037,743; Holland et al., U.S. Pat. No. 5,143,830).

[0056] Methods for introducing exogenous DNA into mammalian host cellsinclude calcium phosphate-mediated transfection, Wigler et al., Cell14:725 (1978); Corsaro and Pearson, Somatic Cell Genetics 7:603, 1981:Graham and Van der Eb, Virology 52:456 (1973), electroporation, Neumannet al., EMBO J. 1:841-845 (1982), DEAE-dextran mediated transfection,Ausubel et al., eds., Current Protocols in Molecular Biology, (JohnWiley and Sons, Inc., NY, 1987), and liposome-mediated transfection,Hawley-Nelson et al., Focus 15:73 (1993); Ciccarone et al, Focus 15:80(1993). The production of recombinant polypeptides in cultured mammaliancells is disclosed, for example, by Levinson et al., U.S. Pat. No.4,713,339; Hagen et al., U.S. Pat. No. 4,784,950; Palmiter et al., U.S.Pat. No. 4,579,821; and Ringold, U.S. Pat. No. 4,656,134. Suitablecultured mammalian cells include the COS-1 (ATCC No. CRL 1650), COS-7(ATCC No. CRL 1651), BHK (ATCC No. CRL 1632), BHK 570 (ATCC No. CRL10314), 293 [ATCC No. CRL 1573; Graham et al., J. Gen. Virol.36:59-72(1977) and Chinese hamster ovary (e.g. CHO-K1; ATCC No. CCL 61)cell lines. Additional suitable cell lines are known in the art andavailable from public depositories such as the American Type CultureCollection, Rockville, Md. In general, strong transcription promotersare preferred, such as promoters from SV-40 or cytomegalovirus. See,e.g., U.S. Pat. No. 4,956,288. Other suitable promoters include thosefrom metallothionein genes (U.S. Pat. Nos. 4,579,821 and 4,601,978 andthe adenovirus major late promoter.

[0057] Drug selection is generally used to select for cultured mammaliancells into which foreign DNA has been inserted. Such cells are commonlyreferred to as “transfectants”. Cells that have been cultured in thepresence of the selective agent and are able to pass the gene ofinterest to their progeny are referred to as “stable transfectants.” Apreferred selectable marker is a gene encoding resistance to theantibiotic neomycin. Selection is-carried out in the presence of aneomycin-type drug, such as G-418 or the like. Selection systems mayalso be used to increase the expression level of the gene of interest, aprocess referred to as “amplification.” Amplification is carried out byculturing transfectants in the presence of a low level of the selectiveagent and then increasing the amount of selective agent to select forcells that produce high levels of the products of the introduced genes.A preferred amplifiable selectable marker is dihydrofolate reductase,which confers resistance to methotrexate. Other drug resistance genes(e.g. hygromycin resistance, multi-drug resistance, puromycinacetyltransferase) can also be used. Alternative markers that introducean altered phenotype, such as green fluorescent protein, or cell surfaceproteins such as CD4, CD8, Class I MHC, placental alkaline phosphatasemay be used to sort transfected cells from untransfected cells by suchmeans as FACS sorting or magnetic bead separation technology.

[0058] Other higher eukaryotic cells can also be used as hosts,including insect cells, plant cells and avian cells. Transformation ofinsect cells and production of foreign polypeptides therein is disclosedby Guarino et al., U.S. Pat. No. 5,162,222; Bang et al., U.S. Pat. No.4,775,624; and WIPO publication WO 94/06463. The use of Agrobacteriumrhizogenes as a vector for expressing genes in plant cells has beenreviewed by Sinkar et al., J. Biosci. (Bangalore) 11:47-58 (1987).Insect cells can be infected with recombinant baculovirus, commonlyderived from Autographa californica nuclear polyhedrosis virus (AcNPV).See, King, L. A. and Possee, R. D., The Baculovirus Expression System: ALaboratory Guide (Chapman & Hall, London); O'Reilly, D. R. et al.,Baculovirus Expression Vectors: A Laboratory Manual (University Press.,New York, Oxford, 1994); and, Richardson, C. D., Ed., BaculovirusExpression Protocols. Methods in Molecular Biology, (Humana Press,Totowa, N.J., 1995). A second method of making recombinant Zcyto10baculovirus utilizes a transposon-based system described by Luckow, V.A, et al., J Virol 67:4566-79 1993). This system, which utilizestransfer vectors, is sold in the Bac-to-Bac™ kit (Life Technologies,Rockville, Md.). This system utilizes a transfer vector, pFastBac™ (LifeTechnologies) containing a Tn7 transposon to move the DNA encoding theZcyto10 polypeptide into a baculovirus genome maintained in E. coli as alarge plasmid called a “bacmid.” See, Hill-Perkins, M. S. and Possee, R.D., J Gen Virol 71:971-6, (1990); Bonning, B. C. et al., J Gen Virol75:1551-6 (1994); and, Chazenbalk, G. D., and Rapoport, B., J Biol Chem270:1543-9 (1995). In addition, transfer vectors can include an in-framefusion with DNA encoding an epitope tag at the C- or N-terminus of theexpressed Zcyto10 polypeptide, for example, a Glu-Glu epitope tag,Grussenmeyer, T. et al., Proc. Natl. Acad. Sci. 82:7952-4 (1985). Usinga technique known in the art, a transfer vector containing Zcyto10 istransformed into E. Coli, and screened for bacmids which contain aninterrupted lacZ gene indicative of recombinant baculovirus. The bacmidDNA containing the recombinant baculovirus genome is isolated, usingcommon techniques, and used to transfect Spodoptera frugiperda cells,e.g. Sf9 cells. Recombinant virus that expresses Zcyto 10 issubsequently produced. Recombinant viral stocks are made by methodscommonly used the art.

[0059] The recombinant virus is used to infect host cells, typically acell line derived from the fall armyworm, Spodoptera frugiperda. See, ingeneral, Glick and Pasternak, Molecular Biotechnology: Principles andApplications of Recombinant DNA, ASM Press, Washington, D.C. (1994).Another suitable cell line is the High FiveO™ cell line (Invitrogen)derived from Trichoplusia ni (U.S. Pat. No. 5,300,435). Commerciallyavailable serum-free media are used to grow and maintain the cells.Suitable media are Sf900 II™ (Life Technologies) or ESF 921™ (ExpressionSystems) for the Sf9 cells; and Ex-cellO405™ (JRH Biosciences, Lenexa,Kans.) or Express FiveO™ (Life Technologies) for the T. ni cells. Thecells are grown up from an inoculation density of approximately 2-5×10⁵cells to a density of 1-2×10⁶ cells at which time a recombinant viralstock is added at a multiplicity of infection (MOI) of 0.1 to 10, moretypically near 3. Procedures used are generally described in availablelaboratory manuals (King, L. A. and Possee, R. D., ibid.; O'Reilly, D.R. et al., ibid.; Richardson, C. D., ibid.). Subsequent purification ofthe Zcyto10 polypeptide from the supernatant can be achieved usingmethods described herein.

[0060] Fungal cells, including yeast cells, and particularly cells ofthe genus Saccharomyces, can also be used within the present invention,such as for producing protein fragments or polypeptide fusions. Methodsfor transforming yeast cells with exogenous DNA and producingrecombinant polypeptides therefrom are disclosed by, for example,Kawasaki, U.S. Pat. No. 4,599,311; Kawasaki et al., U.S. Pat. No.4,931,373; Brake, U.S. Pat. No. 4,870,008; Welch et al., U.S. Pat. No.5,037,743; and Murray et al., U.S. Pat. No. 4,845,075. Transformed cellsare selected by phenotype determined by the selectable marker, commonlydrug resistance or the ability to grow in the absence of a particularnutrient (e.g., leucine). A preferred vector system for use in yeast isthe POT1 vector system disclosed by Kawasaki et al. (U.S. Pat. No.4,931,373), which allows transformed cells to be selected by growth inglucose-containing media. Suitable promoters and terminators for use inyeast include those from glycolytic enzyme genes (see, e.g., Kawasaki,U.S. Pat. No. 4,599,311; Kingsman et al., U.S. Pat. No. 4,615,974; andBitter, U.S. Pat. No. 4,977,092 and alcohol dehydrogenase genes. Seealso U.S. Pat. Nos. 4,990,446; 5,063,154; 5,139,936 and 4,661,454.Transformation systems for other yeasts, including Hansenula polymorpha,Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces fragilis,Ustilago maydis, Pichia pastoris, Pichia methanolica, Pichiaguillermondii and Candida maltosa are known in the art. See, forexample, Gleeson et al., J. Gen. Microbiol. 132:3459-3465 (1986) andCregg, U.S. Pat. No. 4,882,279. Aspergillus cells may be utilizedaccording to the methods of McKnight et al., U.S. Pat. No. 4,935,349.Methods for transforming Acremonium chrysogenum are disclosed by Suminoet al., U.S. Pat. No. 5,162,228. Methods for transforming Neurospora aredisclosed by Lambowitz, U.S. Pat. No. 4,486,533.

[0061] The use of Pichia methanolica as host for the production ofrecombinant proteins is disclosed in WIPO Publications WO 97/17450, WO97/17451, WO 98/02536, and WO 98/02565. DNA molecules for use intransforming P. methanolica will commonly be prepared asdouble-stranded, circular plasmids, which are preferably linearizedprior to transformation. For polypeptide production in P. methanolica,it is preferred that the promoter and terminator in the plasmid be thatof a P. methanolica gene, such as a P. methanolica alcohol utilizationgene (AUG1 or AUG2). Other useful promoters include those of thedihydroxyacetone synthase (DHAS), formate dehydrogenase (FMD), andcatalase (CAT) genes. To facilitate integration of the DNA into the hostchromosome, it is preferred to have the entire expression segment of theplasmid flanked at both ends by host DNA sequences. A preferredselectable marker for use in Pichia methanolica is a P. methanolica ADE2gene, which encodes phosphoribosyl-5-aminoimidazole carboxylase (AIRC;EC 4.1.1.21), which allows ade2 host cells to grow in the absence ofadenine. For large-scale, industrial processes where it is desirable tominimize the use of methanol, it is preferred to use host cells in whichboth methanol utilization genes (AUG1 and AUG2) are deleted. Forproduction of secreted proteins, host cells deficient in vacuolarprotease genes (PEP4 and PRB1) are preferred. Electroporation is used tofacilitate the introduction of a plasmid containing DNA encoding apolypeptide of interest into P. methanolica cells. It is preferred totransform P. methanolica cells by electroporation using an exponentiallydecaying, pulsed electric field having a field strength of from 2.5 to4.5 kV/cm, preferably about 3.75 kV/cm, and a time constant (t) of from1 to 40 milliseconds, most preferably about 20 milliseconds.

[0062] Prokaryotic host cells, including strains of the bacteriaEscherichia coli, Bacillus and other genera are also useful host cellswithin the present invention.

[0063] Techniques for transforming these hosts and expressing foreignDNA sequences cloned therein are well known in the art (see, e.g.,Sambrook et al., ibid.). When expressing a Zcyto10 polypeptide inbacteria such as E. coli, the polypeptide may be retained in thecytoplasm, typically as insoluble granules, or may be directed to theperiplasmic space by a bacterial secretion sequence. In the former case,the cells are lysed, and the granules are recovered and denatured using,for example, guanidine isothiocyanate or urea. The denatured polypeptidecan then be refolded and dimerized by diluting the denaturant, such asby dialysis against a solution of urea and a combination of reduced andoxidized glutathione, followed by dialysis against a buffered salinesolution. In the latter case, the polypeptide can be recovered from theperiplasmic space in a soluble and functional form by disrupting thecells (by, for example, sonication or osmotic shock) to release thecontents of the periplasmic space and recovering the protein, therebyobviating the need for denaturation and refolding.

[0064] Transformed or transfected host cells are cultured according toconventional procedures in a culture medium containing nutrients andother components required for the growth of the chosen host cells. Avariety of suitable media, including defined media and complex media,are known in the art and generally include a carbon source, a nitrogensource, essential amino acids, vitamins and minerals. Media may alsocontain such components as growth factors or serum, as required. Thegrowth medium will generally select for cells containing the exogenouslyadded DNA by, for example, drug selection or deficiency in an essentialnutrient which is complemented by the selectable marker carried on theexpression vector or co-transfected into the host cell. P. methanolicacells are cultured in a medium comprising adequate sources of carbon,nitrogen and trace nutrients at a temperature of about 25° C. to 35° C.Liquid cultures are provided with sufficient aeration by conventionalmeans, such as shaking of small flasks or sparging of fermentors. Apreferred culture medium for P. methanolica is YEPD (2% D-glucose, 2%Bacto™ Peptone (Difco Laboratories, Detroit. Mich.), 1% Bacto™ yeastextract (Difco Laboratories), 0.004% adenine and 0.006% L-leucine).

[0065] Within one aspect of the present invention, a novel protein isproduced by a cultured cell, and the cell is used to screen for areceptor or receptors for the protein, including the natural receptor,as well as agonists and antagonists of the natural ligand.

[0066] Protein Isolation:

[0067] It is preferred to purify the polypeptides of the presentinvention to ≧80% purity, more preferably to ≧90% purity, even morepreferably ≧95% purity, and particularly preferred is a pharmaceuticallypure state, that is greater than 99.9% pure with respect tocontaminating macromolecules, particularly other proteins and nucleicacids, and free of infectious and pyrogenic agents. Preferably, apurified polypeptide is substantially free of other polypeptides,particularly other polypeptides of animal origin.

[0068] Expressed recombinant polypeptides (or chimeric polypeptides) canbe purified using fractionation and/or conventional purification methodsand media. Ammonium sulfate precipitation and acid or chaotropeextraction may be used for fractionation of samples. Exemplarypurification steps may include hydroxyapatite, size exclusion, FPLC andreverse-phase high performance liquid chromatography. Suitable anionexchange media include derivatized dextrans, agarose, cellulose,polyacrylamide, specialty silicas, and the like. PEI, DEAE, QAE and Qderivatives are preferred, with DEAE Fast-Flow Sepharose (Pharmacia,Piscataway, N.J.) being particularly preferred. Exemplarychromatographic media include those media derivatized with phenyl,butyl, or octyl groups, such as Phenyl-Sepharose FF (Pharmacia),Toyopearl butyl 650 (Toso Haas, Montgomeryville, Pa.), Octyl-Sepharose(Pharmacia) and the like; or polyacrylic resins, such as Amberchrom CG71 (Toso Haas) and the like. Suitable solid supports include glassbeads, silica-based resins, cellulosic resins, agarose beads,cross-linked agarose beads, polystyrene beads, cross-linkedpolyacrylamide resins and the like that are insoluble under theconditions in which they are to be used. These supports may be modifiedwith reactive groups that allow attachment of proteins by amino groups,carboxyl groups, sulfhydryl groups, hydroxyl groups and/or carbohydratemoieties. Examples of coupling chemistries include cyanogen bromideactivation, N-hydroxysuccinimide activation, epoxide activation,sulfhydryl activation, hydrazide activation, and carboxyl and aminoderivatives for carbodiimide coupling chemistries. These and other solidmedia are well known and widely used in the art, and are available fromcommercial suppliers. Methods for binding receptor polypeptides tosupport media are well known in the art. Selection of a particularmethod is a matter of routine design and is determined in part by theproperties of the chosen support. See, for example, AffinityChromatography: Principles & Methods (Pharmacia LKB Biotechnology,Uppsala, Sweden, 1988).

[0069] The polypeptides of the present invention can be isolated byexploitation of their properties. For example, immobilized metal ionadsorption (IMAC) chromatography can be used to purify histidine-richproteins. Briefly, a gel is first charged with divalent metal ions toform a chelate (E. Sulkowski, Trends in Biochem. 3:1-7 (1985).Histidine-rich proteins will be adsorbed to this matrix with differingaffinities, depending upon the metal ion used, and will be eluted bycompetitive elution, lowering the pH, or use of strong chelating agents.Other methods of purification include purification of glycosylatedproteins by lectin affinity chromatography and ion exchangechromatography (Methods in Enzymol., Vol. 182, “Guide to ProteinPurification”, M. Deutscher, (ed.), pp. 529-539 (Acad. Press, San Diego,1990. Alternatively, a fusion of the polypeptide of interest and anaffinity tag (e.g., polyhistidine, maltose-binding protein, animmunoglobulin domain) may be constructed to facilitate purification.

[0070] Uses

[0071] The polypeptide of the present invention has the structuralcharacteristics of a four-helix bundle cytokine. A protein is generallycharacterized as a cytokine by virtue of its solubility and ability toact via cell surface receptors to signal and modulate cellproliferation. Cytokines fall into several tertiary structural foldclasses, including cysteine-rich dimers (e.g., insulin, PDGF),beta-trefoil folds (e.g., FGF, IL-1), and all-alpha four helix bundles.The latter are characterized by four helices, labeled A, B, C and D, ina unique up-up-down-down topology, where two overhand loops link helicesA and B and helices C and D. See, for example, Manavalan et al., Journalof Protein Chemistry 11(3): 321-31, (1992). The four-helix bundlecytokines are sometimes further subdivided into short chain (e.g., IL-4,Il-2, GM-CSF) and long chain (e.g., TPO, growth hormone, leptin, IL-10),where the latter generally display longer A and D helices and overhandloops. Henceforth we shall use the term “cytokine” synonymously with“four-helix bundle cytokine”. Helix A of zcyto10 includes amino acidresidue 35, an isoleucine, through amino acid residue 49, an isoleucine,also defined by SEQ ID NO:14; helix B includes amino acid 91, a leucine,through amino acid 105, a threonine, also defined by SEQ ID NO:15; helixC includes amino acid residue 112, a leucine, through amino acid residue126, a cysteine, also defined by SEQ ID NO:16; helix D includes aminoacid residue 158, a valine, through amino acid residue 172, amethionine, also defined by SEQ ID NO:17.

[0072] Human Zcyto10 has an intramolecular disulfide bond between Cys33and Cys126. The other four cysteines, Cys80, Cys132, Cys81 and Cys134are predicted to form two intramolecular disulfide bonds in thearrangement Cys80-Cys132 and Cys8′-Cys134. Residues that are predictedto be crucial for the structural stability of Zcyto10 include Cys33,Cys126, Cys80, Cys132, Cys81 and Cys134. Mutation of any one of theseresidues to any other residue is expected to inactivate the function ofZcyto10.

[0073] The structural stability of Zcyto10 is also dependent on themaintenance of a buried hydrophobic face on the four alpha helices.Residues Ile42, Phe46, Ile49, Leu91, Val94, Phe95, Tyr98, Leu112,Phe116, Ile119, Leu123, Val158, Leu162, Leu165, Leu168, Leu169 andMet172 are predicted to be buried in the core of the protein and if theyare changed, the substituted amino acid residue must be a hydrophobicamino acid.

[0074] Residues expected to be involved in binding of Zcyto10 to a cellsurface receptor include Asp57, on the overhand loop between helix A andB, and Lys160 and Glu164, charged residues predicted to be exposed onthe surface of helix D. On the surface of the protein, on the loop ABand helix D areas, is a hydrophobic surface patch comprising residuesIle62, Leu71, Ile167, and Trp171. These residues may interact with ahydrophobic surface patch on a cell surface receptor.

[0075] The human Zcyto10 polypeptide of the present invention has abouta 28% identity to interleukin-10 (IL-10). Mouse Zcyto10 polypeptide hasapproximately 24% identity to human IL-10, and about 27% identity tomouse IL-10. Human Zcyto10 polypeptide has approximately 76% identitywith mouse Zcyto10 polypeptide.

[0076] Helix A of mouse Zcyto10 includes amino acid residue 35, anisoleucine, through amino acid residue 49, an arginine, of SEQ ID NO:19, also defined by SEQ ID NO:21. Helix B of mouse Zcyto10 includesamino acid residue 91, a leucine, through amino acid residue 105, athreonine, of SEQ ID NO: 19, also defined by SEQ ID NO:22. Helix C ofmouse Zcyto10 includes amino acid residue 112, a leucine, through aminoacid residue 126, a cysteine, of SEQ ID NO: 19, also defined by SEQ IDNO:23. Helix D of mouse Zcyto10 includes amino acid residue 158, avaline, through amino acid residue 172, a methionine, of SEQ ID NO: 19,also defined by SEQ ID NO:24.

[0077] IL-10 is a cytokine that inhibits production of other cytokines,induces proliferation and differentiation of activated B lymphocytes,inhibits HIV-1 replication and exhibits antagonistic effects on gammainterferon. IL-10 appears to exist as a dimer formed from twoalpha-helical polypeptide regions related by a 180° rotation. See, forexample, Zdanov et al., Structure: 3(6): 591-601 (1996). IL-10 has beenreported to be a product of activated Th2 T-cells, B-cells,keratinocytes and monocytes/macrophages that is capable of modulating aTh1 T-cell response. Such modulation may be accomplished by inhibitingcytokine synthesis by Th1 T-cells. See, for example, Hus et al., Int.Immunol. 4: 563 (1992) and D'Andrea et al., J. Exp. Med. 178: 1042(1992). IL-10 has also been reported to inhibit cytokine synthesis bynatural killer cells and monocytes/macrophages. See, for example, Hus etal. cited above and Fiorentino et al., J. Immunol. 146: 3444 (1991). Inaddition, IL-10 has been found to have a protective effect with respectto insulin dependent diabetes mellitus.

[0078] In analysis of the tissue distribution of the mRNA correspondingto this novel DNA, a single transcript was observed at approximately 1.2kb. Using Clontech Multiple Tissue Northerns, the human transcript wasapparent in trachea, placenta, testis, skin, salivary gland, prostate,thyroid with less expression observed in stomach and pancreas. Zcyto10was expressed in the following mouse tissues: kidney, skeletal muscle,salivary gland, liver and skin.

[0079] The tissue specificity of Zcyto10 expression suggests thatZcyto10 may be a growth and/or maintenance factor in the trachea andsalivary glands, stomach, pancreas and muscle; and may be important inlocal immune responses. Also, the Zcyto10 gene's location on chromosome1q32.2 indicates that Zcyto10 is a growth/differentiation factor orimportant in regulating the immune response as IL-10.

[0080] The present invention also provides reagents which will find usein diagnostic applications. A probe comprising the Zcyto10 DNA or RNA ora subsequence thereof can be used to determine if the Zcyto10 gene ispresent on chromosome 1 or if a mutation has occurred.

[0081] The present invention also provides reagents with significanttherapeutic value. The Zcyto10 polypeptide (naturally occurring orrecombinant), fragments thereof, antibodies and anti-idiotypicantibodies thereto, along with compounds identified as having bindingaffinity to the Zcyto10 polypeptide, should be useful in the treatmentof conditions associated with abnormal physiology or development,including abnormal proliferation, e.g., cancerous conditions, ordegenerative conditions or altered immunity.

[0082] Antibodies to the Zcyto10 polypeptide can be purified and thenadministered to a patient. These reagents can be combined fortherapeutic use with additional active or inert ingredients, e.g., inpharmaceutically acceptable carriers or diluents along withphysiologically innocuous stabilizers and excipients. These combinationscan be sterile filtered and placed into dosage forms as bylyophilization in dosage vials or storage in stabilized aqueouspreparations. This invention also contemplates use of antibodies,binding fragments thereof or single-chain antibodies of the antibodiesincluding forms which are not complement binding.

[0083] The quantities of reagents necessary for effective therapy willdepend upon many different factors, including means of administration,target site, physiological state of the patient, and other medicationsadministered. Thus, treatment dosages should be titrated to optimizesafety and efficacy. Typically, dosages used in vitro may provide usefulguidance in the amounts useful for in vivo administration of thesereagents. Animal testing of effective doses for treatment of particulardisorders will provide further predictive indication of human dosage.Methods for administration include oral, intravenous, peritoneal,intramuscular, transdermal or administration into the lung or trachea inspray form by means or a nebulizer or atomizer. Pharmaceuticallyacceptable carriers will include water, saline, buffers to name just afew. Dosage ranges would ordinarily be expected from 11 g to 1000 μg perkilogram of body weight per day. However, the doses by be higher orlower as can be determined by a medical doctor with ordinary skill inthe art. For a complete discussion of drug formulations and dosageranges see Remington's Pharmaceutical Sciences, 18^(th) Ed., (MackPublishing Co., Easton, Penn., 1996), and Goodman and Gilman 's: ThePharmacological Bases of Therapeutics, 9^(th) Ed. (Pergamon Press 1996).

[0084] Nucleic Acid-based Therapeutic Treatment

[0085] If a mammal has a mutated or lacks a Zcyto10 gene, the Zcyto10gene can be introduced into the cells of the mammal. In one embodiment,a gene encoding a Zcyto10 polypeptide is introduced in vivo in a viralvector. Such vectors include an attenuated or defective DNA virus, suchas but not limited to herpes simplex virus (HSV), papillomavirus,Epstein Barr virus (EBV), adenovirus, adeno-associated virus (AAV), andthe like. Defective viruses, which entirely or almost entirely lackviral genes, are preferred. A defective virus is not infective afterintroduction into a cell. Use of defective viral vectors allows foradministration to cells in a specific, localized area, without concernthat the vector can infect other cells. Examples of particular vectorsinclude, but are not limited to, a defective herpes virus I (HSV1)vector [Kaplitt et al., Molec. Cell. Neurosci., 2 :320-330 (1991)], anattenuated adenovirus vector, such as the vector described byStratford-Perricaudet et al., J. Clin. Invest., 90 :626-630 (1992), anda defective adeno-associated virus vector [Samulski et al., J. Virol.,61:3096-3101 (1987); Samulski et al. J. Virol., 63:3822-3828 (1989)].

[0086] In another embodiment, the gene can be introduced in a retroviralvector, e.g., as described in Anderson et al., U.S. Pat. No. 5,399,346;Mann et al., Cell, 33:153 (1983); Temin et al., U.S. Pat. No. 4,650,764;Temin et al., U.S. Pat. No. 4,980,289; Markowitz et al., J. Virol.,62:1120 (1988); Temin et al., U.S. Pat. No. 5,124,263; InternationalPatent Publication No. WO 95/07358, published Mar. 16, 1995 by Doughertyet al.; and Blood, 82:845 (1993). Alternatively, the vector can beintroduced by lipofection in vivo using liposomes. Synthetic cationiclipids can be used to prepare liposomes for in vivo transfection of agene encoding a marker [Felgner et al., Proc. Natl. Acad. Sci. USA,84:7413-7417 (1987); see Mackey et al., Proc. Natl. Acad. Sci. USA,85:8027-8031 (1988)]. The use of lipofection to introduce exogenousgenes into specific organs in vivo has certain practical advantages.Molecular targeting of liposomes to specific cells represents one areaof benefit. It is clear that directing transfection to particular cellsrepresents one area of benefit. It is clear that directing transfectionto particular cell types would be particularly advantageous in a tissuewith cellular heterogeneity, such as the pancreas, liver, kidney, andbrain. Lipids may be chemically coupled to other molecules for thepurpose of targeting. Targeted peptides, e.g., hormones orneurotransmitters, and-proteins such as antibodies, or non-peptidemolecules could be coupled to liposomes chemically. These liposomes canalso be administered in spray form into the lung or trachea by means ofan atomizer or nebulizer.

[0087] It is possible to remove the cells from the body and introducethe vector as a naked DNA plasmid and then re-implant the transformedcells into the body. Naked DNA vector for gene therapy can be introducedinto the desired host cells by methods known in the art, e.g.,transfection, electroporation, microinjection, transduction, cellfusion, DEAE dextran, calcium phosphate precipitation, use of a gene gunor use of a DNA vector transporter [see, e.g., Wu et al., J. Biol.Chem., 267:963-967 (1992); Wu et al., J. Biol. Chem., 263:14621-14624(1988)].

[0088] Zcyto10 polypeptides can also be used to prepare antibodies thatspecifically bind to Zcyto10 polypeptides. These antibodies can then beused to manufacture anti-idiotypic antibodies. As used herein, the term“antibodies” includes polyclonal antibodies, monoclonal antibodies,antigen-binding fragments thereof such as F(ab′)₂ and Fab fragments, andthe like, including genetically engineered antibodies.

[0089] Antibodies are defined to be specifically binding if they bind toa Zcyto10 polypeptide with a K_(a) of greater than or equal to 10⁷/M.The affinity of a monoclonal antibody can be readily determined by oneof ordinary skill in the art (see, for example, Scatchard, ibid.).

[0090] Methods for preparing polyclonal and monoclonal antibodies arewell known in the art (see for example, Sambrook et al., MolecularCloning: A Laboratory Manual, Second Edition (Cold Spring Harbor, N.Y.,1989); and Hurrell, J. G. R., Ed., Monoclonal Hybridoma Antibodies:Techniques and Applications (CRC Press, Inc., Boca Raton, Fla., 1982),which are incorporated herein by reference). As would be evident to oneof ordinary skill in the art, polyclonal antibodies can be generatedfrom a variety of warm-blooded animals such as horses, cows, goats,sheep, dogs, chickens, rabbits, mice, and rats. The immunogenicity of aZcyto10 polypeptide may be increased through the use of an adjuvant suchas Freund's complete or incomplete adjuvant. A variety of assays knownto those skilled in the art can be utilized to detect antibodies whichspecifically bind to Zcyto10 polypeptides. Exemplary assays aredescribed in detail in Antibodies: A Laboratory Manual, Harlow and Lane(Eds.), (Cold Spring Harbor Laboratory Press, 1988). Representativeexamples of such assays include: concurrent immunoelectrophoresis.,radio-immunoassays, radio-immunoprecipitations, enzyme-linkedimmunosorbent assays (ELISA), dot blot assays, inhibition or competitionassays, and sandwich assays.

[0091] Antibodies to Zcyto10 are may be used for tagging cells thatexpress the protein, for affinity purification, within diagnostic assaysfor determining circulating levels of soluble protein polypeptides, andas antagonists to block ligand binding and signal transduction in vitroand in vivo.

[0092] Within another aspect of the present invention there is provideda pharmaceutical composition comprising purified Zcyto10 polypeptide incombination with a pharmaceutically acceptable vehicle. Suchcompositions may be useful for modulating of cell proliferation, celldifferentiation or cytokine production in the prevention or treatment ofconditions characterized by improper cell proliferation, celldifferentiation or cytokine production, as are further discussed herein.Moreover, Zcyto10 polypeptides of the present invention may be used intrachea-specific or tracheobronchial-specific applications, such as inthe maintenance or wound repair of the tracheobronchial epithelium orcells underlying the same, in regulating mucous production or mucocilaryclearance of debris or in treatment of asthma, bronchitis or otherdiseases of the tracheobronchial tract. It is expected that Zcyto10polypeptide would be administered at a dose ranging between the samedoses used for Zcyto10-Fc construct to doses 100-fold higher, dependingupon the stability of Zcyto10 polypeptide. Therapeutic doses of Zcyto10would range from 5 to 5000 μg/kg/day.

[0093] The Zcyto10 polypeptide of the present invention is expressedhighly in salivary gland and trachea and has been found in saliva byWestern blot analysis. The salivary glands synthesize and secrete anumber of proteins having diverse biological functions. Such proteinsfacilitate lubrication of the oral cavity (e.g., mucins and proline-richproteins), remineralization (e.g., statherin and ionic proline-richproteins) and digestion (e.g., amylase, lipase and proteases) andprovide anti-microbial (e.g., proline-rich proteins, lysozyme, histatinsand lactoperoxidase) and mucosal integrity maintenance (e.g., mucins)capabilities. In addition, saliva is a rich source of growth factorssynthesized by the salivary glands. For example, saliva is known tocontain epidermal growth factor (EGF), nerve growth factor (NGF),transforming growth factor-alpha (TGF-α), transforming growthfactor-beta (TGF-β), insulin, insulin-like growth factors I and II(IGF-I and IGF-II) and fibroblast growth factor (FGF). See, for example,Zelles et al., J. Dental. Res. 74(12): 1826-32, 1995. Synthesis ofgrowth factors by the salivary gland is believed to beandrogen-dependent and to be necessary for the health of the oral cavityand gastrointestinal tract.

[0094] Thus, Zcyto10 polypeptides, agonists or antagonists thereof maybe therapeutically useful in the regeneration of the gastrointestinaltract or oral cavity. To verify this presence of this capability inZcyto10 polypeptides, agonists or antagonists of the present invention,such Zcyto10 polypeptides, agonists or antagonists are evaluated withrespect to their ability to break down starch according to proceduresknown in the art. Zcyto10 polypeptides, agonists or antagonists thereofmay be useful in the treatment of asthma and other diseases of thetracheobronchial tract, such as bronchitis and the like, by interventionin the cross-regulation of Th1 and Th2 lymphocytes, regulation ofgrowth, differentiation and cytokine production of other inflammatorycellular mediators, such as eosinophils, mast cells, basophils,neutrophils and macrophages. Zcyto10 polypeptides, agonists orantagonists thereof may also modulate muscle tone in thetracheobronchial tract.

[0095] Zcyto10 polypeptides can also be used to treat a number of skinconditions either systemically or locally when placed in an ointment orcream, for example eczema, psoriasis or dry skin conditions in generalor as related skin attentions. Also the Zcyto10 polypeptide can bedirectly injected into muscle to treat muscle atrophy in the elderly,the sick or the bed-ridden.

[0096] Radiation hybrid mapping is a somatic cell genetic techniquedeveloped for constructing high-resolution, contiguous maps of mammalianchromosomes [Cox et al., Science 250:245-250 (1990)]. Partial or fullknowledge of a gene's sequence allows the designing of PCR primerssuitable for use with chromosomal radiation hybrid mapping panels.Commercially available radiation hybrid mapping panels which cover theentire human genome, such as the Stanford G3 RH Panel and the GeneBridge4 RH Panel (Research Genetics, Inc., Huntsville, Ala.), are available.These panels enable rapid, PCR based, chromosomal localizations andordering of genes, sequence-tagged sites (STSs), and othernonpolymorphic- and polymorphic markers within a region of interest.This includes establishing directly proportional physical distancesbetween newly discovered genes of interest and previously mappedmarkers. The precise knowledge of a gene's position can be useful in anumber of ways including: 1) determining if a sequence is part of anexisting contig and obtaining additional surrounding genetic sequencesin various forms such as YAC-, BAC- or cDNA clones, 2) providing apossible candidate gene for an inheritable disease which shows linkageto the same chromosomal region, and 3) for cross-referencing modelorganisms such as mouse which may be beneficial in helping to determinewhat function a particular gene might have.

[0097] The results showed that the Zcyto10 gene maps 889.26 cR_(—)3000from the top of the human chromosome 1 linkage group on the WICGRradiation hybrid map. Proximal and distal framework markers were D1S504and WI-9641 (D1S2427), respectively. The use of the surrounding markerspositions the Zcyto10 gene in the 1q32.2 region on the integrated LDBchromosome 1 map (The Genetic Location Database, University ofSouthhampton, WWW server:http://cedar.genetics.soton.ac.uk/public_html/). Numerous genes havebeen mapped to the 1q32.2 region of chromosome 1. In particular,mutations in this region have been found to result in van der Woudesyndrome, associated with malformation of the lower lip that issometimes associated with cleft palate. Thus, the Zcyto10 gene, which isexpressed in the salivary gland, may be used in gene therapy of thissyndrome. If a mammal has a mutated or lacks a Zcyto10 gene, the Zcyto10gene can be introduced into the cells of the mammal.

[0098] Another aspect of the present invention involves antisensepolynucleotide compositions that are complementary to a segment of thepolynucleotide set forth in SEQ ID NOs: 1, 3 18 and 33. Such syntheticantisense oligonucleotides are designed to bind to mRNA encoding Zcyto10polypeptides and inhibit translation of such mRNA. Such antisenseoligonucleotides are useful to inhibit expression of Zcyto10polypeptide-encoding genes in cell culture or in a subject.

[0099] The present invention also provides reagents which will find usein diagnostic applications. For example, the Zcyto10 gene, a probecomprising Zcyto 10 DNA or RNA or a subsequence thereof can be used todetermine if the Zcyto10 gene is present on chromosome 1 or if amutation has occurred. Detectable chromosomal aberrations at the Zcyto10gene locus include but are not limited to aneuploidy, gene copy numberchanges, insertions, deletions, restriction site changes andrearrangements. Such aberrations can be detected using polynucleotidesof the present invention by 15 employing molecular genetic techniques,such as restriction fragment length polymorphism (RFLP) analysis, shorttandem repeat (STR) analysis employing PCR techniques, and other geneticlinkage analysis techniques known in the art [Sambrook et al., ibid.;Ausubel, et. al., ibid.; Marian, A. J., Chest, 108: 255-265, (1995)].

[0100] Those skilled in the art will recognize that the sequencesdisclosed in SEQ ID NOs: 2, 4 12, 13, 19, 20, 25, 26, 34 and 35represent a single alleles of the human and mouse Zcyto10 genes andpolypeptides, and that allelic variation and alternative splicing areexpected to occur. Allelic variants can be cloned by probing cDNA orgenomic libraries from different individuals according to standardprocedures. Allelic variants of the DNA sequence shown in SEQ ID NOs: 1,3, 18 and 33 including those containing silent mutations and those inwhich mutations result in amino acid sequence changes, are within thescope of the present invention.

[0101] The sequence of Zcyto10 has 7 message instability motifs in the3′ untranslated region at positions 706, 813, 855 and 906 of SEQ IDNO:1. Treatment of cells expressing Zcyto10 with cycloheximide canalleviate this message instability. See Shaw, G. et. al., Cell 46:659-667 (1986). Furthermore, the AT rich 3′ untranslated region can begenetically altered or removed to further promote message stability.

Use of Zcyto10 to Promote Wound Healing

[0102] The data of Example 4 shows that Zcyto10 plays a role in woundhealing. Thus, Zcyto10 can be applied to a wound or a burn to promotewound healing. Zctyo10 may be administered systemically in a dosage offrom 1 to 100 μg per kilogram weight of the individual. Zcyto10 may alsobe applied to a wound by means of a salve or ointment which containsfrom 1 ng to 1 mg of Zcyto10 to gram of salve or ointment. SeeRemington's Pharmaceutical Sciences, 18^(th) Ed., (Mack Publishing Co.,Easton, Penn., 1996). Zcyto10 should be placed on a cleaned wound on adaily basis until the wound has healed.

Use of Zcyto10 to Increase Platelet Count

[0103] As can be seen below in Example 7, we have discovered thatZcyto10 can be used to increase platelet count. This is especiallyimportant to cancer patients who experience thrombocytopenia due tochemotherapy or radiation therapy. The Zcyto10 can be administeredtherapeutically in with a pharmaceutically acceptable carrier.

[0104] The invention is further illustrated by the followingnon-limiting examples.

EXAMPLE 1 Cloning of Zcyto10

[0105] The full length sequence of zcyto10×1 (the longer form) andzcyto10×2 (the shorter form) was elucidated by using 3′ RACE® andsubmitting two fragments generated to sequencing (SEQ ID NO:10 and SEQID NO:11), then artificially splicing together by computer the estsequence shown in SEQ ID NO:5 with the overlapping sequence from the two3′ race fragments.

[0106] An oligo, zc15907 (SEQ ID NO: 6), was designed to the area justupstream (5′) of the putative methionine for zcyto10. Furtherdownstream, another oligo, zc15906 (SEQ ID NO: 7), was designed to thearea just upstream of the signal sequence cleavage site. These oligoswere used in 3′ RACE reactions on human trachea marathon cDNA. ZC 15907was used in the primary 3′ race reaction and zc 15906 was used in thenested 3′ race reaction. The MARATHON cDNA was made using the MarathoncDNA Amplification Kit (Clontech, Palo Alto, Calif.) according to themanufacturer's instructions, starting with human trachea mRNA purchasedfrom Clontech.

[0107] The PCR reactions were run according to the manufacturer'sinstructions in the Marathon cDNA Amplification Kit with somemodification in the thermal cycling parameters. The cycling parametersused in the primary PCR reaction were:

[0108] 94° C. 1 min 30 sec 1×

[0109] 94° C. 15 sec 68° C. 1 min 30×

[0110] 72° C. 7 min 1×

[0111] The cycling parameters used in the nested PCR reaction were: 94°C. 1 min 30 sec 1×, 94° C. 15 sec 68° C. 1 min 20 sec, 30×72° C. 7 min1×.

[0112] The resulting products were run out on a 1.2% agarose gel (Gibcoagarose) and two main bands were seen, approximately 80 bp apart. Thebands were cut out and gel purified-using QIAEX™ resin (Qiagen)according to the manufacturer's instructions. These fragments were thensubjected to sequencing, allowing the full length sequence of zcyto10 tobe discerned.

EXAMPLE 2 Northern Blot Analysis

[0113] Human multiple tissue blots I, II, III, and a RNA Master Dot Blot(Clontech) were probed to determine the tissue distribution of zcyto10.A 45-mer antisense oligo, SEQ ID NO:9, was designed using the estsequence (SEQ ID NO: 5 bp 100-145) and used for the probe.

[0114] 15 pm of SEQ ID NO: 9 were end labeled with ³²P using T4polynucleotide kinase (Gibco-BRL). The labeling reaction contained 2 μl5× forward kinase reaction buffer (Gibco-BRL), 1 ul T4 kinase, 15 pm SEQID NO:9, 1 ul 6000 Ci/mmol ³²P gamma-ATP (Amersham) and water to 10 ul.The reaction was incubated 30 minutes at 37° C. Unincorporatedradioactivity was removed with a NucTrap Probe Purification Column(Stratagene). Multiple tissue northerns and a human RNA Master Blot(Clontech) were prehybridized at 50° C. three hours in 10 ml ExpressHyb(Clontech) which contained 1 mg of salmon sperm DNA and 0.3 mg humancot1 DNA (Gibco-BRL), both of which were boiled 3 minutes, iced 2minutes and then added to the ExpressHyb. Hybridization was carried outover night at 50 C. Initial wash conditions were as follows: 2×SSC, 0.1%SDS RT for 40 minutes with several wash solution changes, then 1×SSC,0.1% SDS at 64° C. (Tm-10) for 30 minutes. Filters were then exposed tofilm two days.

[0115] Expression of zcyto10 on the northern blots revealed about a 1.2kb band in trachea, a faint 1.5 kb band in stomach and fainter bands ofboth sizes in pancreas. The dot blots showed the presence of zcyto10 intrachea, salivary gland, placenta, testis, skin, prostate gland, adrenalgland and thyroid.

[0116] In the mouse it was found in the kidney, skeletal muscle,salivary gland, liver and skin.

EXAMPLE 3 Chromosomal Assignment and Placement of Zcyto10

[0117] Zcyto10 was mapped to chromosome 1 using the commerciallyavailable version of the “Stanford G3 Radiation Hybrid Mapping Panel”(Research Genetics, Inc., Huntsville, Ala.). The “Stanford G3 RH Panel”contains PCRable DNAs from each of 83 radiation hybrid clones of thewhole human genome, plus two control DNAs (the RM donor and the A3recipient). A publicly available WWW server(http://shgc-www.stanford.edu) allows chromosomal localization ofmarkers.

[0118] For the mapping of Zcyto10 with the “Stanford G3 RH Panel”, 20 μlreactions were set up in a PCRable 96-well microtiter plate (Stratagene,La Jolla, Calif.) and used in a “RoboCycler Gradient 96” thermal cycler(Stratagene). Each of the 85 PCR reactions consisted of 2 μl 10×KlenTaqPCR reaction buffer (CLONTECH Laboratories, Inc., Palo Alto, Calif.),1.6 μl dNTPs mix (2.5 mM each, PERKIN-ELMER, Foster City, Calif.), 1 μlsense primer, SEQ ID NO: 6, 5′ ATT CCT AGC TCC TGT GGT CTC CAG 3′, 1 μlantisense primer, (SEQ ID NO: 8) 5′ TCC CAA ATT GAG TGT CTT CAG T 3′, 2μl “RediLoad” (Research Genetics, Inc., Huntsville, Ala.), 0.4 μl 50×Advantage KlenTaq Polymerase Mix (Clontech Laboratories, Inc.), 25 ng ofDNA from an individual hybrid clone or control and x μl ddH₂O for atotal volume of 20 μl. The reactions were overlaid with an equal amountof mineral oil and sealed. The PCR cycler conditions were as follows: aninitial 1 cycle 5 minute denaturation at 95° C., 35 cycles of a 1 minutedenaturation at 95° C., 1 minute annealing at 66° C. and 1.5 minuteextension at 72° C., followed by a final 1 cycle extension of 7 minutesat 72° C. The reactions were separated by electrophoresis on a 2%agarose gel (Life Technologies, Gaithersburg, Md.).

[0119] The results showed linkage of Zcyto10 to the framework makerSHGC-36215 with a LOD score of >10 and at a distance of 14.67cR_(—)10000from the marker. The use of surrounding markers positions Zcyto10 in the1q32.2 region on the integrated LDB chromosome 1 map (The GeneticLocation Database, University of Southhampton, WWW server:http://cedar.genetics.soton.ac.uk/public html/).

EXAMPLE 4 Use of Zcyot10 to Promote Wound Healing

[0120] Normal adult female Balb/C mice were used in the present study.They were housed in animal care facilities with a 12-hour light-darkcycle, given water and laboratory rodent chow ad libitum during thestudy. They were individually caged from the day of surgery.

[0121] On the day of surgery, the animals were anesthetized withketamine (Vetalar, Aveco Inc., Ft. Dodge, IA) 104 mg/kg plus Xylazine(Rompun, Mobey Corp., Shawnee, Kans.) 7 mg/kg in sterile (0.2μ-filtered) phosphate buffered saline (PBS) by intraperitonealinjection. The hair on their backs was clipped and the skin depilatedwith NAIR® (Carter-Wallace, New York, N.Y.), then rinsed with water.100% aloe vera gel was applied to counteract the alkaline bum from theNAIR® treatment, then the animals were placed on circulating waterheating pads until the skin and surrounding fur were dry.

[0122] The animals were then anesthetized with metofane (Pittman Moore,Mundelein, N.J.) and the depilated dorsum wiped with 70% ethanol. Fourexcisions, each of 0.5-cm square were made through the skin andpanniculus camosus over the paravertebral area at the level of thethoracic-lumbar vertebrae. The wounds and surrounding depilated skinwere covered with an adhesive, semipermeable occlusive dressing,BIOCLUSIVE® (Johnson & Johnson, Arlington, Tex.). The cut edge of theexcision was traced through the BIOCLUSIVE® onto an acetate transparencyfor later assessment of closure parameters.

[0123] Control skin and wounded skin at different time points (7 hours,15 hours and 24 hours) were processed using the Qiagen RNeasy Midi kit.Briefly, skin (control and wounded areas) were weighed and homogenizedin appropriate volume of lysis buffer (RLT). The lysates were spun toremove tissue debris and equal volume of 70% ethanol was added to thelysates; mixed well and loaded on column. The samples were spun fiveminutes and washed once with 3.8 ml of RW1 buffer, then twice with 10RPE (2.5 ml each). The total RNA's were eluted with RNase-free water.The expression level of the skin samples were measured using real timePCR (Perkin Elmer ABI Prism 7700 Sequence Detector).

[0124] The experiment was designed with a non template control, a set ofstandard and the skin samples. Mouse kidney total RNA was use for thestandard curve. Three sets of skin total RNA's (25 ng) were used in thisexperiment 7 hours (control and wounded); 15 hours (control andwounded), 24 hours (control and wounded). Each sample was done intriplicate by One Step RT-PCR on the 7700 sequence detector. Thein-house forward primer SEQ ID NO:36, reverse primer SEQ ID NO:37, andthe Perkin Elmer's TaqMan probe (ZG-7-FAM) were used in the experiment.The condition of the One Step RT-PCR was as follow: (RT step) 48° C. for30 minutes, (40 cycles PCR step) 95° C. for 10 minutes, 95° C. for 15second, 60° C. for 1 minute.

[0125] The expression level of cyto10 in the control skin samples at 7hours and 15 hours were comparable at 2.46 ng/ml and 2.61 ng/mlrespectively. From the control skin sample at 24 hours, the expressionlevel of Zcyto10 was zero. The expression level of cyto10 from woundedskin at 7 hours was at 5.17 ng/ml (more than two fold increase comparedto that of the control sample). The expression level of cyto10 fromwounded skin at 15 hours was at 14.45 ng/ml (5.5 fold increase comparedto that of the control sample). The expression level of cyto10 fromwounded skin at 24 hours was at 5.89 ng/ml. A repeat experiment alsoincluded a negative control (yeast tRNA) gave the similar trend and theresult of yeast tRNA was near zero. The result suggested that theamplification was real and mouse specific.

[0126] These data suggest that Zcyto10 plays a role in the repair ofwounded because the expression level of Zcyto10 from wounded tissue wasup compared to that of the control sample and it increased and decreasedafter time. Thus, Zcyto 10 can be applied to wounds to promote woundhealing.

EXAMPLE 5 Transgenic Mice

[0127] Transgenic mice were produced which expressed Zcyto10 eitherunder the albumin or the metallothionine promoter. At birth, several ofthe mice had a shiny appearance and had limited movement. The skin ofthese mice was tight and wrinkled, several also had a whisker-like hairon the lower lip. The nostril and mouth areas, the extremities and thetail were swollen.

[0128] One transgenic mouse, in which the albumin promoter was usedsurvived until day three and was severely growth retarded. There was noear 15 development and the development of the toes was diminished. Allanimals were sacrificed when they were moribund on days 1, 2 or 3. Tailsand liver samples were collected and they were fixed in situ in 10%neutral formalin embedded in paraffin, and sectioned at 3 micrometersand stained with H&E. All mice with this phenotype were transgenic andhad low to high expression of Zcyto10.

[0129] No significant changes were observed in the majority of thetissues except for the skin. The skin of the zcyto10 expressing pups,particularly the those mice which had a high expression level of Zcyto10, tended to be thicker than the non-expressing pups. The stratumgranulosum in these pups appeared to be reduced in thickness as comparedto the non-expressing pups, while the stratum spinosum was thicker dueto increased cell layers and/or increased cell diameter.

[0130] In addition to the changes in the epidermis, the dermis of onemouse having medium expression of Zcyto 10 was focally moderatelyexpanded by mucinous material.

EXAMPLE 6 Purification of Zcyto10 from a Cell Culture Medium

[0131] Zcyto10 produced by CHO cells was isolated from the cell culturemedium using a two step method involving a cation exchangechromatography and size exclusion chromatography.

[0132] A. Cation Exchange Chromatography Step.

[0133] Materials Used

[0134] 2.2 cm diameter (D)×6 cm height (H) column (AMICON) packed with aSP-650M cation exchange resin, which is a TOYOPEARL ion exchange resinhaving covalently bonded sulfopropyl (SP) groups.

[0135] Fifteen (15) liters of culture medium from baby hamster kidney(BHK) cells which had been transfected with a Zcyto10 containing plasmidwas collected. The pH of the culture medium was adjusted to pH5 with 2NHCl. The above-described packed column was equilibrated with 50 mMsodium acetate, NaAc, pH5.0. The culture medium was loaded onto thecolumn at the rate of 20 column-volumes (cv)/hr at approximately 8ml/min. When the loading was done the column was washed with 10 cv of 50mM NaAc, pH5.0. The material in the column was then eluted with 20 cv ofNaCl gradient in 50 mM NaAc, pH 5.0. The NaCl gradient ranged from 0 to0.5 M NaCl. This concentrated the material in the culture medium from 15liters to 170 ml.

[0136] The resultant 170 ml harvest was further concentrated to about 5ml with a spin 5 thousand cut-off centrifugal concentrator (Millipore,Inc. Bedford, Mass.).

[0137] B. Size Exclusion (S-100) Gel Filtration Step

[0138] Materials Used.

[0139] Column 1.6 cm (diameter)×93 cm (height)

[0140] S-100 gel (Pharmacia, Piscataway, N.J.)

[0141] The 5 ml harvest was then loaded onto the above-described columncontaining S-100 gel. The column had been equilibrated with 5× phosphatebuffered saline to bring the pH of the column to about 7.0. Zcyto10 wasisolated from the contaminants by using 1×PBS at a flow rate of 1.5ml/min. Fractions were collected at 2 ml increments. The Zcyto10polypeptide came out in fractions 52-64 at about 90 minutes after theelution had been initiated as determined by sodium dodecyl sulfate (SDS)polyacrylamide gel electrophoresis which were stained with CoomassieBlue. The gel revealed one band at the predicted molecular weight ofabout 14 kDa.

EXAMPLE 7 Cloning of Murine Zcyto10

[0142] PCR primers 5′ MARATHON RACE™ (Clontech, Palo Alto, Calif.)primer set SEQ ID NO: 38 attached to MARATHON™ API adapter, nested withSEQ ID NO:39 attached to AP2 MARATHON™ adapter, with 3′ MARATHON RACE™primer set SEQ ID NO: 40 attached to MARATHON RACE™ API adapter, nestedwith SEQ ID NO:41 attached to MARATHON RACE™ AP2 adapter and 5′ and 3′race was performed on mouse skin MARATHON RACE™ cDNA. Several fragmentswere from these reactions were gel purified and sequenced, allowing theelucidation of the full length coding sequence of the mouse zcyto10,plus some 5′ and 3′ UTR sequence. Two murine Zcyto10 variants werediscovered, namely SEQ ID NOs: 18 and 19 and SEQ ID NOs: 33 and 34. Theclones were amplified by PCR using primers SEQ ID NOs:42 and 43.

EXAMPLE 8 Adenovirus Administration of Zcyto10 to Normal Mice

[0143] Zcyto10 was administered by adenovirus containing the Zcyto10gene. There were three groups of mice as described below. The adenoviruswas injected intravenously into C57B1/6 male and female mice. All micereceived bromodeoxyuridine (BrdU) in their drinking water 3 days beforesacrifice. This allowed for detection of cell proliferation byhistologic methods. Parameters measured included weight change, completeblood counts, serum chemistries, histology, organ weights and cellproliferation by BrdU.

[0144] Experimental Design Group 1 Zcyto10X1 (SEQ ID NO: 18)/pAC-CMV/AdV1 × 10¹¹ particles/dose (9 females, 9 males sacrificed on day 21) (2females, 2 males sacrificed on day 11) total number = 22 mice. Group 2null CMV/AdV control 1 × 10¹¹ particles/dose (10 females, 10 malessacrificed on day 21) (2 females, 2 males sacrificed on day 11) totalnumber = 24 mice. Group 3 no treatment (5 females, 5 males) total number= 10.

[0145] Results

[0146] The most striking effect was a significant increase in plateletcount which was observed in male and female mice treated withZcyto10-adenovirus compared to empty adenovirus control. This wasaccompanied in male mice by a decrease hematocrit and increased spleenand liver weight. The thymus weight was decreased in males also. Incontrast Zcyto10-adenovirus treated female mice showed significantlyincreased white blood cell counts which were consisted primarily ofincreased lymphocyte and neutrophil counts compared to the empty viruscontrol.

[0147] These results suggest that hematopoiesis is effected by Zcyto10treatment, but except for the increased platelet count which effectedboth sexes, other effects are sex specific.

[0148] Other Effects Included the Following.

[0149] Female glucose levels were lower in treated groups while those ofthe males showed no significant change.

[0150] Blood Urea Nitrogen (BUN) was higher in both male and femaletreated groups.

[0151] Female alkaline phosphatase was higher in the treated group whilethe males showed no significant change.

What is claimed is:
 1. An expression vector comprising the followingoperably linked elements: (a) a transcription promoter; (b) a DNAsegment encoding a polypeptide that is at least 90% identical in aminoacid sequence to residues 25 to 176 of SEQ ID NO:2; and (c) atranscription terminator.
 2. The expression vector according to claim 1,further comprising a secretory signal sequence operably linked to theDNA segment.
 3. The expression vector according to claim 1, wherein thepolypeptide comprises an affinity tag or an immunoglogulin constantregion.
 4. An expression vector comprising the following operably linkedelements: (a) a transcription promoter; (b) a DNA segment encoding apolypeptide comprising amino acid residues 25 to 176 of SEQ ID NO:2; and(c) a transcription terminator.
 5. The expression vector according toclaim 4, further comprising a secretory signal sequence operably linkedto the DNA segment.
 6. The expression vector according to claim 4,wherein the polypeptide comprises an affinity tag or an immunoglogulinconstant region.
 7. An expression vector comprising the followingoperably linked elements: (a) a transcription promoter; (b) a DNAsegment encoding a polypeptide comprising SEQ ID NO:2; and (c) atranscription terminator.
 8. The expression vector according to claim 7,further comprising a secretory signal sequence operably linked to theDNA segment.
 9. The expression vector according to claim 7, wherein thepolypeptide comprises an affinity tag or an immunoglogulin constantregion.
 10. An expression vector comprising the following operablylinked elements: (a) a transcription promoter; (b) a DNA segmentencoding a polypeptide comprising amino acid residues 25 to 151 of SEQID NO:4; and (c) a transcription terminator.
 11. The expression vectoraccording to claim 10, further comprising a secretory signal sequenceoperably linked to the DNA segment.
 12. The expression vector accordingto claim 10, wherein the polypeptide comprises an affinity tag or animmunoglogulin constant region.
 13. An expression vector comprising thefollowing operably linked elements: (a) a transcription promoter; (b) aDNA segment encoding polypeptide comprising SEQ ID NO:4; and (c) atranscription terminator.
 14. The expression vector according to claim13, further comprising a secretory signal sequence operably linked tothe DNA segment.
 15. The expression vector according to claim 13,wherein the polypeptide comprises an affinity tag or an immunoglogulinconstant region.
 16. An expression vector comprising the followingoperably linked elements: (a) a transcription promoter; (b) a DNAsegment encoding a polypeptide comprising SEQ ID NO:13; and (c) atranscription terminator.
 17. The expression vector according to claim16, further comprising a secretory signal sequence operably linked tothe DNA segment.
 18. The expression vector according to claim 18,wherein the polypeptide comprises an affinity tag or an immunoglogulinconstant region.
 19. An expression vector comprising the followingoperably linked elements: (a) a transcription promoter; (b) a DNAsegment encoding a polypeptide comprising SEQ ID NO:26; and (c) atranscription terminator.
 20. The expression vector according to claim19, further comprising a secretory signal sequence operably linked tothe DNA segment.
 21. The expression vector according to claim 19,wherein the polypeptide comprises an affinity tag or an immunoglogulinconstant region.
 22. An expression vector comprising the followingoperably linked elements: (a) a transcription promoter; (b) a DNAsegment encoding a polypeptide comprising SEQ ID NO:19; and (c) atranscription terminator.
 23. The expression vector according to claim22, further comprising a secretory signal sequence operably linked tothe DNA segment.
 24. The expression vector according to claim 22,wherein the polypeptide comprises an affinity tag or an immunoglogulinconstant region.
 25. An expression vector comprising the followingoperably linked elements: (a) a transcription promoter; (b) a DNAsegment encoding a polypeptide comprising SEQ ID NO:25; and (c) atranscription terminator.
 26. The expression vector according to claim26, further comprising a secretory signal sequence operably linked tothe DNA segment.
 27. The expression vector according to claim 26,wherein the polypeptide comprises an affinity tag or an immunoglogulinconstant region.
 28. An expression vector comprising the followingoperably linked elements: (a) a transcription promoter; (b) a DNAsegment encoding a polypeptide comprising amino acid residues 25 to 154of SEQ ID NO:34; and (c) a transcription terminator.
 29. The expressionvector according to claim 28, further comprising a secretory signalsequence operably linked to the DNA segment.
 30. The expression vectoraccording to claim 28, wherein the polypeptide comprises an affinity tagor an immunoglogulin constant region.
 31. The expression vectoraccording to claim 28, wherein the polypeptide comprises SEQ ID NO:34.32. A cultured cell into which has been introduced the expression vectoraccording to claim 1, wherein said cell expresses the polypeptideencoded by the DNA segment.
 33. A cultured cell into which has beenintroduced the expression vector according to claim 4, wherein said cellexpresses the polypeptide encoded by the DNA segment.
 34. A culturedcell into which has been introduced the expression vector according toclaim 7, wherein said cell expresses the polypeptide encoded by the DNAsegment.
 35. A cultured cell into which has been introduced theexpression vector according to claim 13, wherein said cell expresses thepolypeptide encoded by the DNA segment.
 36. A cultured cell into whichhas been introduced the expression vector according to claim 16, whereinsaid cell expresses the polypeptide encoded by the DNA segment.
 37. Acultured cell into which has been introduced the expression vectoraccording to claim 19, wherein said cell expresses the polypeptideencoded by the DNA segment.
 38. A cultured cell into which has beenintroduced the expression vector according to claim 22, wherein saidcell expresses the polypeptide encoded by the DNA segment.
 39. Acultured cell into which has been introduced the expression vectoraccording to claim 25, wherein said cell expresses the polypeptideencoded by the DNA segment.
 40. A cultured cell into which has beenintroduced the expression vector according to claim 28, wherein saidcell expresses the polypeptide encoded by the DNA segment.
 41. A methodof producing a polypeptide comprising: culturing a cell into which hasbeen introduced the expression vector according to claim 32, whereby thecell expresses the polypeptide encoded by the DNA segment; and isolatingthe polypeptide produced by the cell.
 42. A method of producing apolypeptide comprising: culturing a cell into which has been introducedthe expression vector according to claim 33, whereby the cell expressesthe polypeptide encoded by the DNA segment; and isolating thepolypeptide produced by the cell.
 43. A method of producing apolypeptide comprising: culturing a cell into which has been introducedthe expression vector according to claim 34, whereby the cell expressesthe polypeptide encoded by the DNA segment; and isolating thepolypeptide produced by the cell.
 44. A method of producing apolypeptide comprising: culturing a cell into which has been introducedthe expression vector according to claim 35, whereby the cell expressesthe polypeptide encoded by the DNA segment; and isolating thepolypeptide produced by the cell.
 45. A method of producing apolypeptide comprising: culturing a cell into which has been introducedthe expression vector according to claim 36, whereby the cell expressesthe polypeptide encoded by the DNA segment; and isolating thepolypeptide produced by the cell.
 46. A method of producing apolypeptide comprising: culturing a cell into which has been introducedthe expression vector according to claim 37, whereby the cell expressesthe polypeptide encoded by the DNA segment; and isolating thepolypeptide produced by the cell.
 47. A method of producing apolypeptide comprising: culturing a cell into which has been introducedthe expression vector according to claim 38, whereby the cell expressesthe polypeptide encoded by the DNA segment; and isolating thepolypeptide produced by the cell.
 48. A method of producing apolypeptide comprising: culturing a cell into which has been introducedthe expression vector according to claim 39, whereby the cell expressesthe polypeptide encoded by the DNA segment; and isolating thepolypeptide produced by the cell.
 49. A method of producing apolypeptide comprising: culturing a cell into which has been introducedthe expression vector according to claim 40, whereby the cell expressesthe polypeptide encoded by the DNA segment; and isolating thepolypeptide produced by the cell.